Parasitic diseases caused by members of the trypanosome family threaten five hundred million people worldwide and are responsible for an estimated 30 million infections at any time, representing an enormous public health and economic toll in affected regions of the world. These diseases include African trypanosomiasis (sleeping sickness), American trypanosomiasis (Chagas'disease) and leishmaniasis. African trypanosomiasis is resurgent after having been contained with significant success in the first part of the 20th century;Chagas'disease has spread beyond the original geographic boundaries of Latin America due to patterns of immigration and now threatens the U.S., and leishmaniasis has moved from an exotic disease to one which has been brought home at increasing frequency by personnel returning to the U.S. from the Mideast. To address the technical as well as operational needs for better and more accurate parasite detection and identification, we propose to develop a molecular identification assay that can be performed in a modest laboratory environment by personnel with corresponding level of training. The trypanosome reverse line blot (Tryp-RLB) assays to be developed will comprise two sequential procedures: Polymerase chain reaction amplification using primers derived from conserved regions within the parasite genomes, followed by hybridization against species, subspecies or strain-specific probes arrayed on a membrane. The membrane will be prepared with oligonucleotides covalently attached in a macroarray and packaged in a disposable multichannel incubation cassette, permitting parallel hybridization of multiple PCR-amplified samples per assay run. The Tryp-RLB assay will be carried out in a flow-through aspiration device which both accelerates the assay turnaround time and prevents contamination by collecting amplification products in a sealed flask. Same day assay results are obtained, a considerable advantage over both culture-based and many other molecular methods. The Tryp-RLB assay to be developed herein for identification of trypanosome and Leishmania species and strains can be applied to a wide variety of problems in areas of epidemiology and diagnostics as well as therapeutics, vaccines and other basic research. Its format allows it to be both generic in procedure and application specific in choice of target sequences for the membrane macroarray, providing a powerful analytical tool which is without precedent in the field of trypanosome biology. PUBLIC HEALTH RELEVANCE: Human African trypanosomiasis, American trypanosomiasis (Chagas'disease) and leishmaniasis directly affect over 30 million people in endemic regions of the Americas, Asia and Africa, while ten times that number are at risk of infection. Diagnosis and epidemiological monitoring of these diseases has been challenging due to the complex life cycles of the parasitic agents causing them, the difficulty in identifying symptoms of infection, and the cross-reactivities between many pathogen species. This project will result in the development of a molecular assay for detection and identification of the parasitic species and strains responsible for both forms of trypanosomiasis and leishmaniasis. Earlier and more accurate detection and identification than is possible with current methods will enable clinicians to select more effective antimicrobial therapy for patients, and will contribute to epidemiological monitoring and surveillance of the spread of these parasitic agents.